Enteric pathogenic E. coli assay For the detection of EAEC, EPEC, EIEC and ETEC genes using the BD MAX TM system. Instructions for use (Version 1.0 March 2017) Contents 1
Introduction Contact information 1. Protocol 1.1. Materials required 1.2. Run settings 1.3. Sample preparation 1.4. Setting up the experiment 2. Result interpretation 3. Data of Test-Validation 2
Introduction Enteric pathogenic E. coli comprise a group of distinct E. coli pathotypes equipped with special virulence factors, which enables these E. coli strains to colonize the human gut and cause intestinal disease (diarrhoea). The respective pathotypes, enteroaggregativen (EAEC), enteropathogenic (EPEC), enteroinvasive (EIEC) and enterotoxigenic (ETEC) E. coli can be differentiated and detected by their distinct virulence gene repertoire. The specific virulence genes are detected with this multiplex PCR assay as described below. This protocol describes the system settings and setup protocols for running the EP-2 panel to detect four pathogens using the BD MAX TM system (assay target genes): 1. EAEC - aggr 2. EPEC eae and EAF 3. EIEC - ipah 4. ETEC - LT The qpcr has been primarily validated for bacterial culture from agar plates, e.g. MacConkey agar. However, using DNA extraction systems appropriate for stool samples allows the qpcr test directly from the patient s stool. Contact information For information regarding ordering dried snap-in tubes for the EP-2 assay: info@biolegio.com For information regarding to the protocol: schubert@med.uni-muenchen.de 3
1. Protocol This protocol describes the assay settings required for running the EP-2 assay on the BD MAX TM system. The EP-2 snap-in assay contains primers and probes for the detection of EAEC, EPEC, EIEC and ETEC. Materials needed BD MAX TM system BD MAX TM ExK DNA-2 Extraction Kit (BD cat.no: 442820) or BD MAX TM ExK DNA-4 Extraction Kit (BD cat.no: 442850) BD MMK Mastermix with SPC (BD cat.no: 442829) BD MAX TM PCR Cartridges (BD cat.no: 437519) Dried snap-ins EP-2 (Biolegio cat no: BDT-14016) Eppendorftubes 1,5 ml Vortex Mixer Micropipettes Safeseal Filtertips Disposable gloves Inoculating loop (10 µl) 1.1 Run settings The assay is performed on the BD MAX TM system with use of the BD MMK with SPC in combination with the ExK DNA-2 Kit or ExK DNA-4 Kit for the extraction. Create a full process assay in the test editor named EP-2 assay, use the corresponding Extraction type 2 or 4 (as shown in the screenshot of Basic Information ) and the following parameters: 4
or ExK DNA 4 Edit the test steps using the following settings: 5
1.3. Sample preparation a) Bacterial culture Swab of bacteria cultivated on agar plate (e.g. MacConkey agar) dispensed in 1000 µl A. dest Prepare a 1:100 Dilution Transfer 200 µl of the 1:100 diluted sample into a BD MAX TM DNA Sample Buffer Tube and close the tube with a blue septum cap. Ensure complete mixing by vortexing the sample Extraction will be done with the BD MAX TM ExK DNA-2 or ExK DNA-4 Extraction Kit 6
1.4. Setting up the experiment a. Create a worklist on the BD MAX instrument using the EP-2 assay (created in step 1.2) and label the lanes with the sample names. b. Load the prepared Sample Buffer Tubes into their corresponding position in the BD MAX racks. c. Load the BD MAX racks with the corresponding Unitized Reagent Strip. d. Snap in the BD Extraction tubes (position 1), MMK tubes (position 2) and EP-2 tubes (position 3) into the Reagent Strip. e. Load the racks and cartridges into the BD MAX and Start Run 2. Result interpretation 2.1 For a run to be valid No BD MAX System failures. Negative Control (optional) has a Cq value of -1 for all channels Positive control (optional) has a Cq value for channel 475/520, 530/565, 585/ 630, 630/665 and 680/715 2.2 Interpretation if run is valid A Cq value of -1 indicates a negative result A Cq value for either of the targets indicates a positive result for the corresponding target. The SPC (channel 680/715) should always give a Cq value, if there is no other target positive. A negative value for the SPC together with negative Cq value in every other channel indicates inhibition. Therefore this sample should be repeated. All curves need to be visually checked for right interpretation. 7
3. Validation 3.1 Targets 3.1.1. EAEC: aggr (GenBank Acc. No.: Z32523) 3.1.2. EPEC: eae and EAF (GenBank Acc.No.: AB040740/ X76137) 3.1.3. EIEC: ipah (GenBank Acc. No.: M32063) 3.1.4. ETEC: LT (GenBank Acc. No.: S60731) 3.2. Sensitivity / Specificity / Analytical Sensitivity The sensitivity and specificity was determined using 15 defined positive and 25 defined negative samples, which were unequivocally assessed positive and negative by 2 independent other PCRs, respectively. The analytical sensitivity was determined by log-step dilutions of bacterial strains (CFU) carrying the respective DNA target sequences. All PCRs have been used in our clinical microbiological diagnostic laboratory for the last 8 years under accreditation. They having passed constantly external inter-laboratory comparison programmes (once a year). Target analytical sensitivity (bacterial culture) EAEC 10 4 copies/ ml EPEC 10 4 copies/ ml EIEC 10 2 copies/ ml ETEC 10 3 copies/ ml Disclaimer: MvP-Institut is not responsible for the results on the EP-2 assay on the BD MAX system. Using the open protocol, the respective laboratory itself is responsible for the validation of the assay. 8